Coactivators and Androgen Receptors in Prostate Cancer

描述(由申请人提供)： 前列腺癌最初是一种雄激素依赖性疾病，越来越多的证据表明雄激素受体(AR)在雄激素消融抵抗前列腺癌中继续发挥作用。近年来，在阐明AR的结构、确定候选的共调节蛋白以及确定一些受AR调控的基因方面取得了长足的进步。尽管如此，我们对AR作用机制的了解以及AR调控的诱导前列腺癌细胞生长的关键基因的识别知识还不足以为合理开发阻断AR活性的替代方法提供良好的基础。我们对雄激素非依赖性前列腺癌的了解和有效阻止其生长的能力甚至还不够先进。虽然许多候选的AR共激活子已经通过分子生物学技术被发现，但几乎不知道它们在细胞中的个体作用，它们对AR靶基因的正负调控以及对AR依赖的细胞生长的贡献。此外，它们在雄激素非依赖性AR作用中的作用尚未确定。为了解决这些问题，我们建议：具体目标1：验证雄激素受体在某些雄激素非依赖性前列腺癌细胞和雄激素依赖性前列腺癌细胞的生长和基因表达中起关键作用的假设，并评估受体共激活因子在激素依赖和非激素依赖的细胞生长和基因调控中的作用。我们将选择性地减少候选共激活因子的表达，并评估其对细胞生长和靶基因表达的影响。具体目的2：确定雄激素受体调控的基因，并验证辅激活子要求是靶基因/启动子特异性的假设。我们将利用两种方法，Affymetrix U133A芯片和一种新的含有TEV(烟草蚀刻病毒)蛋白酶位点连接子的蛋白A-AR嵌合体来识别AR靶基因，并评估辅活化子减少对靶基因表达的影响。具体目的3：利用染色质免疫沉淀(ChIP)方法鉴定雄激素受体参与激素依赖和配体非依赖性转录调控的协同调节因子，并评估特定协同调节因子的消除对启动子相关蛋白的组成和启动子相关蛋白的翻译后修饰的影响。

DESCRIPTION (provided by applicant): Prostate cancer is initially an androgen dependent disease and there is mounting evidence that the androgen receptor (AR) continues to play a role in androgen ablation resistant prostate cancer. Great strides have been made in recent years in elucidating the structure of AR, identifying candidate coregulatory proteins, and in identifying some of the genes regulated by AR. Nonetheless, our understanding of the mechanism of action of AR and our knowledge of the identity of the key genes regulated by AR to induce growth of prostate cancer cells is insufficient to provide a good basis for rational development of alternate means of blocking AR activity. Our understanding of and ability to effectively block the growth of androgen independent prostate cancer is even less well advanced. Although many candidate AR coactivators have been identified by molecular biological techniques, virtually nothing is known about their individual roles in cells, their contributions to positive and negative regulation of AR target genes and to AR dependent cell growth. Moreover, their role in androgen independent AR action has not been determined. To address these issues, we propose: Specific Aim 1: To test the hypothesis that the androgen receptor plays a key role in the growth and gene expression in some androgen independent prostate cancer cells as well as in androgen dependent prostate cancer cells and to evaluate the role of receptor coactivators in both hormone dependent and independent cell growth and gene regulation. We will selectively reduce expression of candidate coactivators and assess the effects on cell growth and target gene expression. Specific Aim 2: To identify genes regulated by androgen receptor and to test the hypothesis that coactivator requirements are target gene/promoter specific. We will utilize two approaches, Affymetrix U133A chips and a novel Protein A-AR chimera containing a TEV (tobacco etch virus) protease site linker to identify AR target genes and to evaluate the consequences of coactivator reduction on target gene expression. Specific Aim 3: To utilize chromatin immunoprecipitation (CHIP) assays to identify coregulators involved in hormone dependent and ligand independent transcriptional regulation by androgen receptors and to assess the consequences of elimination of specific coregulators on the composition of proteins associated with promoters and on the post-translational modifications of proteins associated with the promoter.